It is often desirable to detect very small amounts of extracted or in vitro amplified nucleic acids, for example in biological samples. According to the most common approach, the target nucleic acid is hybridized to an oligonucleotide. In order to obtain a detectable signal, proportionate to the amount of the target, either the target nucleic acid or the oligonucleotide needs to be associated with a signal generating reporter element, such as a radioactive atom or a chromogenic molecule, or, an enzyme such as alkaline phosphatase. The signal generated by a properly hybridized nucleic acid can be detected and measured by methods known in the art. Many of the commonly used techniques of molecular biology require the immobilization of the targets on solid supports, to enable fractionation and identification of specific sequences. The target nucleic acid may be captured by oligonucleotides immobilized on solid supports, or more frequently, so-called "sandwich" hybridization systems are employed, using a capture oligonucleotide covalently attached to a solid support for capturing detection oligonucleotide-target nucleic acid adducts formed in solution. Typical solid supports are, for example, nitrocellulose or nylon membranes, activated agarose supports or diazotized cellulose supports. However, the bonds between these supports and the oligonucleotides are either not covalent, thereby allowing a certain release of the oligonucleotides from the support, or the supports have other shortcomings. For example, N-hydroxysuccinimide or cyanogen bromide activated polysaccharide affinity supports have a serious drawback in the leakage of ligands. This not only leads to misleading results but, even more importantly, poses health hazards when immunoaffinity-purified products produced by recombinant DNA synthesis are complexed with mouse monoclonal antibodies [see e.g. Wilchek et al., Biochemistry 26, 2155 (1987) and Wilchek et al. PNAS 72, 1055 (1975)]. Leakage from solid support obviously interferes with affinity purification: if the free ligand that leaks from the support is more effective as a binder than the insolubilized ligand, the free ligand will bind the target macromolecule essentially irreversibly, and prevent affinity adsorption to the column. Further, cyanogen bromide activation of polysaccharide supports leads to the formation of N-substituted isoureas on the surface of the matrix. These confer undesirable ion exchange properties to the support, which become problematic in affinity chromatography, when analytes (such as nucleic acids) are present in very minute concentrations.
The attachment of oligonucleotides containing an aldehyde or carboxylic acid group at the 5'-terminus to non-porous polystyrene latex solid microspheres is disclosed in Kremsky et al., Nucleic Acids Research 15, 2891 (1987). Although this method provides good end-attachment results, it is disadvantageous in that at the end of the coupling reaction, non-covalently bound oligonucleotide requires removal by a tedious gel electrophoresis step.
Therefore, solid supports with cross-linked, porous polymeric matrix structures that are able to capture and covalently bind oligonucleotides are preferred. For example Sephacryl beads are widely used due to their excellent hybridization properties.
A so called "bead-based sandwich hybridization system" (BBSHS) is, for example, described in the following publications: EP 276, 302 and Gingeras et al., PNAS (in press). According to this method, in a first step, a target nucleic acid and an oligonucleotide probe used for its detection, which is complementary to at least a region of the target, are hybridized. The obtained adduct is then captured by a second oligonucleotide, that is complementary to a different region of the target, and is end-attached to a solid support. The amount of the detection oligonucleotide associated with the solid support is directly related to the amount of the target captured. In this way, the BBSHS can be used to determine the amount of a single-stranded nucleic acid in a sample. In this and similar assays most commonly radioactively (e.g., .sup.32 P) labeled cloned DNAs or synthetic oligonucleotides are employed. .sup.32 P-labeled oligonucleotide probes used in conjunction with Sephacryl.TM. dextran beads in BBSHS experiments provide about 10:1 or better signal to noise ratios with target sequences present in about 0.5 fmole amounts.
In practice, non-radioisotopic reporter systems are often preferred, primarily due to the inconveniences associated with handling, storage and disposal of radioisotopes. Successful application of a non-radioisotopic reporter system requires a detection system which exhibits high sensitivities and low background properties when used in conjunction with the reporter system on a given solid support. The Sephacryl.TM. dextran beads supports show serious limitations when used in conjunction with non-radioisotopic, e.g. colorimetric, detection systems. For example, the colorimetric signal from enzyme-oligonucleotide conjugates in sandwich formats and direct capture experiments on Sephacryl.TM. dextran beads was compromised by undesirable background, thereby giving low signal to noise ratios. In the presence of target, the non-specific background can be a result of:
1) hybridization of the detection and capture oligonucleotide to non-exact sequences of the target nucleic acid;
2) hybridization of the detection oligonucleotide to the capture oligonucleotide;
3) non-specific attachment of the detection oligonucleotide to the bead support or walls of the reaction vessel.
While the first two of these possible causes can be minimized by sufficiently stringent solution hybridization, capture and wash conditions, the reasons for non-specific binding properties are poorly understood.
It would be desirable to find solid supports that have better binding properties, e.g. on which the non-specific attachment of the oligonucleotides used for detection of the target nucleic acids is lower and which show a greater capture potential of the immobilized probe, especially when used in conjunction with nonradioisotopic detection systems.
The properties most sought for in solid supports used for detection of nucleic acids are:
hydrophilicity PA0 ease of handling, such as compatibility with centrifugation techniques PA0 the presence of suitable functional groups PA0 low non-specific binding of the detection oligonucleotides.
In search for new solid supports, attention focused on polyacrylamide-based matrices. These supports are commercially available in a wide range of pore sizes, and are used routinely, for example, in affinity chromatography. Their hydrophilicity, lack of charged residues on their surface, and ease of derivatization are some of the properties which make them potentially attractive as supports for the attachment of oligonucleotides. Chemical derivatization of these cross-linked polyacrylamide beads for use in affinity chromatography provided spaced-out functional groups for attaching specific ligands in orientations favorable for specific binding with various macromolecules, thereby enabling the selective retention of these macromolecules, e.g., proteins [Inman, J. K., Meth. Enzymol. 34:30 (1974)]. Inman presented a convenient method for the preparation of hydrazide derivatives of cross-linked polyacrylamides by reacting their primary amide groups with hydrazine. Depending on the reaction conditions, hydrazide derivatives with different levels of hydrazide functionality were obtained. The hydrazide derivatives are suitable starting materials for the preparation of other derivatives. For example, bromoacetyl-derivatized polyacrylamide matrices can be obtained by reacting the hydrazide derivatives with N-hydroxysuccinimide ester of bromoacetic acid, on the analogy of the reaction described by Bernatowicz et al., Anal. Biochem. 155, 95 (1986).
Our further goal was to develop a methodology for the attachment of oligonucleotides to solid supports by which the nucleic acids are tethered to the solid supports by their 5'-ends. In the case of the conventionally used solid supports, e.g. Sephacryl.TM. dextran beads, the degree of end-attachment is relatively low (about 50-55%; Ghosh et al., Nucl. Acids Res. 15:5353; (1987)). A higher degree of end-attachment would be manifested in greater capture potential of the immobilized oligonucleotide probe.
As an object of the present invention, it has been found that thiol and bromoacetyl groups of suitably derivatized oligonucleotides and polyacrylamide solid supports react with reasonable yields and their reaction, surprisingly, results in an almost 100% end-attachment of the oligonucleotides. Accordingly, thiol-derivatized oligonucleotides were used in combination with bromoacetyl-derivatized polyacrylamide solid supports. Thiol-oligonucleotides can, for example, be prepared as described by Peng Li et al., Nucl. Acids Res. 15, 5275 (1987) or Chu et al., Nucl. Acids Res. 14, 5591 (1986).
Alternatively, bromoacetyl-oligonucleotides were attached to thiol-derivatized polyacrylamide supports. Bromoacetyl-derivatized oligonucleotides are new compounds. The thiol-derivatized polyacrylamide supports are known in the art, and are, for example disclosed in Meth. Enzymol. 34:30 (1974). The known thiol-affinity supports were prepared by the coupling of carboxyl groups with cystamine, followed by reduction with excess DTT and not via hydrazide derivatives as in the process of the present invention.